A Potential Lead for Insect Growth Regulator: Design, Synthesis, and Biological Activity Evaluation of Novel Hexacyclic Pyrazolamide Derivatives

Ecdysone receptor (EcR) and chitinase play a critical role in the molting stage of insect pests. Each of them is considered a promising target for the development of novel insect growth regulators (IGRs). In the present paper, a total of 24 (23 novel) hexacyclic pyrazolamide derivatives were designed and synthesized by reducing the heptacycle and inserting small flexible linkers on the basis of the previously discovered dual-target compound D-27 acting simultaneously on EcR and Ostrinia furnacalis chitinase (OfChtI). Their insecticidal activities against Plutella xylostella, Spodoptera frugiperda, and Ostrinia furnacalis larvae were evaluated. The results revealed that the insecticidal activity was not significantly enhanced when the heptacycle on the pyrazole ring was reduced to a hexacycle. However, the insertion of an additional methylene spacer between the substituted phenyl ring and the amide bond can improve the insecticidal activity. Among the derivatives, the most potent compound, 6j, exhibited promising insecticidal activities against P. xylostella and S. frugiperda. Further protein binding assays and molecular docking indicated that 6j could target both EcR and OfChtI, and is a potential lead compound for IGRs. The present work provides valuable clues for the development of new dual-target IGRs.


Introduction
Insect growth regulators (IGRs) are regarded as a class of ideal pesticides widely used for the control of pest insects, such as mosquitoes, cockroaches, and various Lepidoptera [1][2][3][4]. Compared with conventional insecticides, IGRs offer numerous advantages, such as relative nontoxicity to mammals, decreased incidence of resistance, higher selectivity, and safer eco-toxicological profiles [5][6][7]. They can target specific proteins associated with molting or metamorphosis to induce a range of abnormal growth responses in insects, from deforming or damaging post-ecdysis to failing to develop into larvae or pupae [5,8]. As molting is essential for insect growth and development, ecdysone receptor (EcR) and chitinase, involved in disturbing the process of insect molting and exfoliation, have been indicated as potential targets for the design of highly efficient and low-toxicity green insecticides [9][10][11]. Therefore, ecdysone analogues and chitinase inhibitors are considered as potential IGRs.
Previously, some non-steroidal dibenzoylhydrazine (DBH) IGRs ( Figure 1) have been commercialized [12,13]. Nevertheless, the practical application of these DBHs is limited by their single chemical structure skeleton and resistance [7,14,15]. A number of chitinase inhibitors have been reported to date, such as pentoxifylline, theophylline, caffeine, dimethylguanylurea, etc. [16]. However, no commercial chitinase inhibitors have been reported [16]. Therefore, using effective strategies to design IGRs with simpler structures and higher activity is of great value. A strategy involving the insertion of small flexible linkers is an important scaffold hopping manipulation, which has been successfully employed in the discovery of new pesticides [17]. In our previous work, we found a dual-target compound, D-27, could act on both EcR and Ostrinia furnacalis chitinase (OfChtI), and the molecular hydrophobicity and flexibility play an important role on the binding affinity of ligand with the EcR or OfChtI [6,18]. However, how the length of the bridge chain effects the activity is unclear. Meanwhile, it is a useful effort to discover a better IGR candidate with simpler structure and lower cost.
Therefore, in the present work, a series of compounds (Scheme 1) were designed and synthesized based on the dual-target compound D-27 by the strategy of replacing the heptacyclic ring with a smaller hexacyclic ring in order to simplify the structure and reduce the cost, as well as by involving the insertion of one or two methylene spacers to lengthen the bridge chain and improve molecular flexibility. The biological activities against P. xylostella, S. frugiperda, and O. nubilalis, and the protein binding assays of the compounds, were evaluated and a promising dual target compound, 6j, with better activity than D-27, was hereby found.  In our previous work, we found a dual-target compound, D-27, could act on both EcR and Ostrinia furnacalis chitinase (Of ChtI), and the molecular hydrophobicity and flexibility play an important role on the binding affinity of ligand with the EcR or Of ChtI [6,18]. However, how the length of the bridge chain effects the activity is unclear. Meanwhile, it is a useful effort to discover a better IGR candidate with simpler structure and lower cost.
Therefore, in the present work, a series of compounds (Scheme 1) were designed and synthesized based on the dual-target compound D-27 by the strategy of replacing the heptacyclic ring with a smaller hexacyclic ring in order to simplify the structure and reduce the cost, as well as by involving the insertion of one or two methylene spacers to lengthen the bridge chain and improve molecular flexibility. The biological activities against P. xylostella, S. frugiperda, and O. nubilalis, and the protein binding assays of the compounds, were evaluated and a promising dual target compound, 6j, with better activity than D-27, was hereby found.
Previously, some non-steroidal dibenzoylhydrazine (DBH) IGRs ( Figure 1) have been commercialized [12,13]. Nevertheless, the practical application of these DBHs is limited by their single chemical structure skeleton and resistance [7,14,15]. A number of chitinase inhibitors have been reported to date, such as pentoxifylline, theophylline, caffeine, dimethylguanylurea, etc. [16]. However, no commercial chitinase inhibitors have been reported [16]. Therefore, using effective strategies to design IGRs with simpler structures and higher activity is of great value. A strategy involving the insertion of small flexible linkers is an important scaffold hopping manipulation, which has been successfully employed in the discovery of new pesticides [17]. In our previous work, we found a dual-target compound, D-27, could act on both EcR and Ostrinia furnacalis chitinase (OfChtI), and the molecular hydrophobicity and flexibility play an important role on the binding affinity of ligand with the EcR or OfChtI [6,18]. However, how the length of the bridge chain effects the activity is unclear. Meanwhile, it is a useful effort to discover a better IGR candidate with simpler structure and lower cost.
Therefore, in the present work, a series of compounds (Scheme 1) were designed and synthesized based on the dual-target compound D-27 by the strategy of replacing the heptacyclic ring with a smaller hexacyclic ring in order to simplify the structure and reduce the cost, as well as by involving the insertion of one or two methylene spacers to lengthen the bridge chain and improve molecular flexibility. The biological activities against P. xylostella, S. frugiperda, and O. nubilalis, and the protein binding assays of the compounds, were evaluated and a promising dual target compound, 6j, with better activity than D-27, was hereby found. Scheme 1. Design strategy of the target compounds. Scheme 1. Design strategy of the target compounds.

Insecticidal Biological Activity
The insecticidal activities of the target compounds 6a~6x against lepidoptera agricultural pests P. xylostella, S. frugiperda, and O. nubilalis were preliminarily evaluated, and the corresponding mortalities for pests were presented in Table 1. The commercial insecticide tebufenozide was selected as a positive control. The length of the bridge chain between the amide bond and the substituted phenyl ring is denoted by "n". Where n = 0, compound with no methylene spacer; n = 1, compound with a methylene spacer; n = 2, compound with two methylene spacers.
(n = 0) or with two methylene spacers (n = 2). For instance, 6i (R = 3-Br, n = 1) > 6t (R = 3-Br, n = 2); 6a (R = 3-Br, n = 0), 6j (R = 4-Br, n = 1) > 6b (R = 4-Br, n = 0); and 6u (R = 4-Br, n = 2) (Figure 3a). When n = 1, compounds with fluorine, bromine, methyl, or trifluoromethoxy substituents on the para-position of the benzene ring displayed much better insecticidal activities than those on the meta-position; for instance, 6l (R = 4-OCF3, n = 1) > 6k (R = 3-OCF3, n = 1) and 6n (R = 4-CH3, n = 1) > 6m (R = 3-CH3, n = 1). Additionally, the appearance of the P. xylostella larvae treated with 6j demonstrated some obvious symptoms of abnormal molting and malformation, such as fuscous, hindgut, coarse cuticula, and abnormal molting, similar to those induced with tebufenozide ( Figure 3b). However, the size and color of the heads of P. xylostell treated with 6j were different from those treated with tebufenozide ( Figure 3b), which indicated that compound 6j may act on the molt-related protein receptors, resulting in the abnormal molting of larvae.   Additionally, the appearance of the P. xylostella larvae treated with 6j demonst some obvious symptoms of abnormal molting and malformation, such as fuscous, gut, coarse cuticula, and abnormal molting, similar to those induced with tebufen ( Figure 3b). However, the size and color of the heads of P. xylostell treated with 6j different from those treated with tebufenozide (Figure 3b), which indicated that pound 6j may act on the molt-related protein receptors, resulting in the abnormal mo of larvae.  Additionally, the appearance of the P. xylostella larvae treated with 6j demonstrated some obvious symptoms of abnormal molting and malformation, such as fuscous, hindgut, coarse cuticula, and abnormal molting, similar to those induced with tebufenozide ( Figure 3b). However, the size and color of the heads of P. xylostell treated with 6j were different from those treated with tebufenozide (Figure 3b), which indicated that compound 6j may act on the molt-related protein receptors, resulting in the abnormal molting of larvae.
Furthermore, the growth and development of the S. frugiperda larvae treated w were dramatically inhibited, and their appearance also demonstrated some ob symptoms of abnormal molting and malformation similar to those induced with te nozide (Figure 4b). However, the color of S. frugiperda treated with 6j was different that of those treated with tebufenozide (Figure 4b), which indicated that 6j may h different mode of action from that of tebufenozide. For O. furnacalis, most of the target compounds exhibited low insecticidal activ a concentration of 200 mg/L. To our satisfaction, the potential compound 6j still disp 33.5% mortality against O. furnacalis. Based on the structure-activity study, we found the compounds exhibited better insecticidal activities when n = 1 (Figure 5a). More as depicted in Figures 5b, the growth of the O. furnacalis larvae treated with 6j was gr inhibited and its appearance also showed some obvious symptoms of abnormal mo and malformation, such as extrusion of the hindgut and cuticle fold, which was simi that induced by tebufenozide (Figure 5b). However, the hair color of specimen tr with 6j seems to be darker than that of those treated with tebufenozide. Furthermore, the growth and development of the S. frugiperda larvae treated with 6j were dramatically inhibited, and their appearance also demonstrated some obvious symptoms of abnormal molting and malformation similar to those induced with tebufenozide ( Figure 4b). However, the color of S. frugiperda treated with 6j was different from that of those treated with tebufenozide (Figure 4b), which indicated that 6j may have a different mode of action from that of tebufenozide.
For O. furnacalis, most of the target compounds exhibited low insecticidal activity at a concentration of 200 mg/L. To our satisfaction, the potential compound 6j still displayed 33.5% mortality against O. furnacalis. Based on the structure-activity study, we found that the compounds exhibited better insecticidal activities when n = 1 (Figure 5a). Moreover, as depicted in Figure 5b, the growth of the O. furnacalis larvae treated with 6j was greatly inhibited and its appearance also showed some obvious symptoms of abnormal molting and malformation, such as extrusion of the hindgut and cuticle fold, which was similar to that induced by tebufenozide (Figure 5b). However, the hair color of specimen treated with 6j seems to be darker than that of those treated with tebufenozide.

The Binding Affinity to EcR and OfChtI
The growth and development of P. xylostella, S. frugiperda, and O. furnacalis treated with 6j showed the symptoms of abnormal molting and malformation, similar to those treated with tebufenozide. Previously, D-27 was found to have moderate binding activity to EcR/USP and excellent inhibitory activity to Of ChtI [6,18]. Therefore, in order to clarify the target protein of 6j, the EcR from P. xylostella and chitinase from O. furnacalis were selected for target verification. The binding affinity of D-27 and 6j to EcR/USP was measured with a competitive binding assay using [ 3 H] PonA as the radioactive ligand. The results revealed that 6j had good binding activity (85.5%) at the concentration of 40 mg/L, which was better than that of D-27 (32.5%), but slightly lower than that of tebufenozide (93.7%) (Figure 6a). The inhibiting activity of 6j to Of ChtI was assayed by a microplate reader using 4-methylumbelliferyl−N, N -diacetyl-β-d-chitobioside [4-MU-(GlcNAc) 2 ] as a fluorescent substrate. The obtained results in Figure 6b indicated that the inhibition rate of 6j (66.0%) was comparable to that of lead D-27 (67.6%) at 40 mg/L, but slightly lower than that of the control (GlcN) 5 (75.6%). On the basis of these findings, 6j was considered as a potential dual-target insect growth regulator lead compound, due to its ability to act on both EcR/USP and Of ChtI receptors.

The Binding Affinity to EcR and OfChtI
The growth and development of P. xylostella, S. frugiperda, and O. furnacalis tr with 6j showed the symptoms of abnormal molting and malformation, similar to treated with tebufenozide. Previously, D-27 was found to have moderate binding ac to EcR/USP and excellent inhibitory activity to OfChtI [6,18]. Therefore, in order to c the target protein of 6j, the EcR from P. xylostella and chitinase from O. furnacalis selected for target verification. The binding affinity of D-27 and 6j to EcR/USP was m ured with a competitive binding assay using [ 3 H] PonA as the radioactive ligand results revealed that 6j had good binding activity (85.5%) at the concentration of 40 m which was better than that of D-27 (32.5%), but slightly lower than that of tebufen (93.7%) (Figure 6a). The inhibiting activity of 6j to OfChtI was assayed by a micro reader using 4-methylumbelliferyl−N, N′-diacetyl-β-d-chitobioside [4-MU-(GlcNAc)2 fluorescent substrate. The obtained results in Figure 6b indicated that the inhibition of 6j (66.0%) was comparable to that of lead D-27 (67.6%) at 40 mg/L, but slightly l than that of the control (GlcN)5 (75.6%). On the basis of these findings, 6j was consid as a potential dual-target insect growth regulator lead compound, due to its ability on both EcR/USP and OfChtI receptors.
; n represents the number of methylene spacers in the bridge chain between the amide bond and the substituted benzene ring. Data represent the mean ± standard deviation. A one-way analysis of variance (ANOVA) was performed followed by Duncan s test (Different letters indicate values that are significantly different (p < 0.05)); (b) the abnormal molting and malformation of O. furnacalis larvae after 96 h (From left to right: 6j, tebufenozide, and negative control).

The Binding Affinity to EcR and OfChtI
The growth and development of P. xylostella, S. frugiperda, and O. furnacalis treated with 6j showed the symptoms of abnormal molting and malformation, similar to those treated with tebufenozide. Previously, D-27 was found to have moderate binding activity to EcR/USP and excellent inhibitory activity to OfChtI [6,18]. Therefore, in order to clarify the target protein of 6j, the EcR from P. xylostella and chitinase from O. furnacalis were selected for target verification. The binding affinity of D-27 and 6j to EcR/USP was measured with a competitive binding assay using [ 3 H] PonA as the radioactive ligand. The results revealed that 6j had good binding activity (85.5%) at the concentration of 40 mg/L, which was better than that of D-27 (32.5%), but slightly lower than that of tebufenozide (93.7%) (Figure 6a). The inhibiting activity of 6j to OfChtI was assayed by a microplate reader using 4-methylumbelliferyl−N, N′-diacetyl-β-d-chitobioside [4-MU-(GlcNAc)2] as a fluorescent substrate. The obtained results in Figure 6b indicated that the inhibition rate of 6j (66.0%) was comparable to that of lead D-27 (67.6%) at 40 mg/L, but slightly lower than that of the control (GlcN)5 (75.6%). On the basis of these findings, 6j was considered as a potential dual-target insect growth regulator lead compound, due to its ability to act on both EcR/USP and OfChtI receptors.

Molecular Docking
In order to gain insights into the possible molecular mechanisms of hexacyclic pyrazolamide derivatives binding with EcR and Of ChtI, 6f and D-27 were selected as representative compounds with which to study the effect of the modification of heptacycle into hexacycle on insecticidal activity. Thereafter, 6j and D-27 were selected as representative compounds to reveal the reason why 6j was more effective than the lead D-27 in terms of insecticidal activity. Meanwhile, 6b, 6j, and 6u were selected as representative ligands with which to explore the relationship between the length of the bridge chain and the binding affinities.
Our previous study suggested hydrogen bonding interactions and a hydrophobic effect play an important role in the design of ecdysone analogs; in particular, the hydrogen bond formed by small molecule with Tyr408 is essential for the inhibitory activity. When 6f (hexacyclic) and D-27 (heptacyclic) were docked into the EcR pocket, both of them showed a similar binding mode, by forming hydrogen bonds with Tyr408 and Asn504 ( Figure S1a). Interestingly, 6j and D-27 also displayed similar binding modes to that of tebufenozide in the active site of EcR (Figure 7a), in which all three were able to form hydrogen bonds with Tyr408 and Asn504. However, the distance of the hydrogen bond formed by the carbonyl oxygen of the amide bond with Tyr408 in compound 6j was 3.0 Å (Figure 7c), shorter than that of D-27 (3.2 Å) (Figure 7b), but longer than that of tebufenozide (2.8 Å) (Figure 7d), which may explain the phenomenon that the binding activity of 6j was stronger than that of D-27, but weaker than that of tebufenozide. The differences in the length of the bridge chain between the amide bond and the benzene ring of the same substituent could also affect the binding interactions. For example, 6b (n = 0, R = 4-Br) and 6u (n = 2, R = 4-Br) exhibited similar binding modes to 6j (n = 1, R = 4-Br), but both could only form hydrogen bonding interactions with Asn504, and not with Tyr408 ( Figure S2a).
bond formed by small molecule with Tyr408 is essential for the inhibitory activity. When 6f (hexacyclic) and D-27 (heptacyclic) were docked into the EcR pocket, both of them showed a similar binding mode, by forming hydrogen bonds with Tyr408 and Asn504 ( Figure S1a). Interestingly, 6j and D-27 also displayed similar binding modes to that of tebufenozide in the active site of EcR (Figure 7a), in which all three were able to form hydrogen bonds with Tyr408 and Asn504. However, the distance of the hydrogen bond formed by the carbonyl oxygen of the amide bond with Tyr408 in compound 6j was 3.0 Å (Figure 7c), shorter than that of D-27 (3.2 Å) (Figure 7b), but longer than that of tebufenozide (2.8 Å) (Figure 7d), which may explain the phenomenon that the binding activity of 6j was stronger than that of D-27, but weaker than that of tebufenozide. The differences in the length of the bridge chain between the amide bond and the benzene ring of the same substituent could also affect the binding interactions. For example, 6b (n = 0, R = 4-Br) and 6u (n = 2, R = 4-Br) exhibited similar binding modes to 6j (n = 1, R = 4-Br), but both could only form hydrogen bonding interactions with Asn504, and not with Tyr408 ( Figure S2a). Additionally, the docking of ligands with OfChtI was carried out. The results exhibited that 6f and D-27 had similar binding modes in the OfChtI pocket, and both can interact Additionally, the docking of ligands with Of ChtI was carried out. The results exhibited that 6f and D-27 had similar binding modes in the Of ChtI pocket, and both can interact with Trp107 by hydrogen bond and with Trp34, Phe309, Tyr272, and Trp107 through additional π−π interactions ( Figure S1b). As presented in Figure 8a, superimposed conformations of selected compounds with Of ChtI indicated that the binding mode of 6j was similar to those of the substrate (GlcN) 5 and the lead D-27. Specifically, D-27 could form π−π interactions with Trp34, Phe309, and Tyr272, as well as an additional hydrogen bond with Trp107 (Figure 8b), which can also be found in the docking mode of 6j with Of ChtI (Figure 8c). However, unlike D-27, 6j did not form π−π interactions with Trp107, but formed additional π−π interactions with the key amino acid Trp372 (Figure 8d). When R = 4-Br, the binding modes of compounds 6b (n = 0, R = 4-Br) and 6u (n = 2, R = 4-Br) were consistent with that of 6j (n = 1, R = 4-Br), and all of them could form hydrogen bonds with Trp107 and π−π interaction with Trp372. However, 6j could form stronger interactions with the hydrophobic residues (Phe309 and Tyr272) than 6b and 6u ( Figure S2b). This may be why compound 6j had higher insecticidal activity than 6b and 6u.

General Procedure for the Synthesis of the Intermediates 3~5 and 6a~6x
The synthetic route is depicted in Scheme 2. The spectra and high-resolution mass spectrometry (HRMS) of target compounds 6a~6x are depicted in the Supplementary Materials.

Synthetic Procedure of Intermediate 3
Intermediate 3 was synthesized according to a previously described method, with minor modifications (Scheme 2) [18]. To a 0 • C solution of sodium ethoxide (0.037 mol) in 100 mL of dry ethanol, cyclohexanone (0.074 mol) and diethyl oxalate (0.074 mol) were added dropwise. The mixture was stirred for 6 h at room temperature, then acidified (pH 4-5, with 20% H 2 SO 4 ) and filtered to remove the formed precipitates. The filtrate was extracted with dichloromethane, dried, and concentrated under vacuum, then purified by column chromatography (petroleum ether/ethyl acetate 30:1) to yield the β-keto esters 3 as a red viscous liquid (11.02 g; 75% yield). 1

Synthetic Procedure of Intermediate 4
The key intermediate 4 was prepared by the cyclization of the β-keto ester 3 with phenylhydrazine hydrochloride [6]. To a mixture of phenylhydrazine hydrochloride (0.026 mol) in ethanol (50 mL), triethylamine (TEA) was added to adjust the pH to 7. A solution of the β-keto esters 3 (0.026 mol) in ethanol (15 mL) was added dropwise. The mixture was stirred for 12 h at room temperature. TLC (petroleum ether/ethyl acetate 10:1) monitored the completion of the reaction. The reaction mixture was concentrated under vacuum, and the residue was purified by column chromatography (petroleum ether/ethyl acetate 10:1) to yield the intermediate 4 as a red solid (4.08 g; 58 yield). 1

Synthetic Procedure of Intermediate 5
Pyrazole carboxylic acid 5 was obtained by the saponification reaction of intermediate 4 according to the literature reporting method [12,21]. To a solution of 6 mol/L NaOH(aq) (20 mL), intermediate 4 (3.41 g) was added and the mixture was heated at 80 • C. The reaction was stirred for 2 h and monitored TLC (petroleum ether/ethyl acetate 6:1). After the completion of hydrolysis, ice water (60 mL) was added and the mixture was acidified (pH 2-3) with concentrated HCl. The formed solid was collected by filtration and dried by hot lamp to yield 5 as a white solid (2.77 g; 90% yield). 1

General Procedures for the Preparation of Compounds 6a~6x [6]
The target compound 6a was synthesized as follows. To a solution of 1-hydroxybenzotriazole (HOBt) (2.48 mmol) and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) (2.48 mmol) in dichloromethane (40 mL), the intermediate 5 (2.06 mmol) was added at room temperature. A solution of 3-bromoaniline (2.48 mmol) in dichloromethane (10 mL) was added dropwise. The mixture was stirred overnight at room temperature and monitored by TLC (petroleum ether/ethyl acetate 4:1). After the completion of the reaction, the mixture was washed with dilute hydrochloric acid, saturated sodium bicarbonate solution, and saturated brine, dried with anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate 4:1) to yield the target compound 6a.
The synthesis of 6b~6x are similar to that of 6a. The structures of 6a~6x were corroborated by 1 HNMR, 13 C NMR, and HRMS. All of the physical data and the HPLC chromatogram of 6j are presented in the Supplementary Materials.

Biological Assay
P. xylostella, O. furnacalis, and S. frugiperda were reared continuously with fresh cabbage, corn leaves, and artificial feed, respectively, in our laboratory without insecticides. All target compounds were dissolved in 1 mL of dimethyl sulfoxide and diluted with an aqueous solution of 0.05% (w/v) Triton X-100 to prepare a 500 mg/L working solution. Other test concentrations were serially diluted with an aqueous solution of 0.05% (w/v) Triton X-100. The commercial insecticide tebufenozide was selected as the positive control. Water containing 0.05% (w/v) Triton X-100 with and without dimethyl sulfoxide were used as the negative and blank controls, respectively. The corrected mortality rate calculated as follows: Corrected mortality rate (%) = (T − C) × 100/(100% − C) where T is the mortality rate in the group of tested compounds and C is the mortality rate in the blank control group.

Bioactivity Assay against P. xylostella and O. furnacalis
The insecticidal activities of the target compounds and the control insecticide tebufenozide against P. xylostella and O. furnacalis were conducted using the reported leaf-dipping method [22][23][24][25]. Cabbage or corn leaves were cut and dipped into the test solution for 10 s. After air drying, the treated leaf disks were placed in the Petri dish (10 cm in diameter). Each dry-treated leaf disk was infested with 15 second-instar larvae of P. xylostella or O. furnacalis. Subsequently, the experimental groups were cultivated in an incubator at 27 ± 1 • C, 70 ± 20% RH (relative humidity) and a 14:10 h light/dark photoperiod. The mortality rates were recorded at 96 h. The tests were replicated three times for each treatment (15 larvae per replicate).

Bioactivity Assay against S. frugiperda
The biological activity against S. frugiperda was evaluated as previously described [19,26]. Artificial feed was added to the 24-well plate and 100 µL of the target compound solution was evenly dripped onto the surface of the feed after it had cooled and formed. After drying at room temperature, one second-instar larva was inserted into each well. The concentrations were repeated three times for each group (12 larvae per replicate). Each experiment was performed three times. The 24-well culture plates were stored in an incubator at 26 • C and 85% relative humidity at 16:8 h light/dark photoperiod. The mortality rates were confirmed after 144 h.

Molecular Docking
Molecular docking was performed by using the Surflex-Dock algorithm in Sybyl 7.3 software [27,28]. The crystal structures of the EcR/USP heterodimer (EcR/USP-LBD) from Heliothis virescens (PDB ID: 3IXP) and group I chitinase from O. furnacalis (PDB ID: 3WL1) obtained from the PDB protein data bank were used for the docking study. The docking results were visualized by Pymol (version 1.9.0) (http://www.pymol.org/ accessed on 10 February 2023) and Discovery Studio 2016 Client (for 2D interaction).

Ligand-Inhibiting Assay on OfChtI
Chitinase Of ChtI was expressed and purified according to our published literature [18]. The inhibition assay was carried out following the standard strategy. 4-Methylumbelliferyl β-D-N,N -diacetylchitobioside hydrate [4MU-(GlcNAc) 2 ] (Sigma, Shanghai, China) was used as the substrate to calculate the inhibiting activity on Of ChtI based on fluorescence intensity, as in a previously reported method [11].

Conclusions
In conclusion, a total of 24 (23 novel) hexacyclic pyrazolamide derivatives were designed and synthesized through the strategy of reducing the heptacyclic ring and inserting small flexible linkers. The synthetic route was easy to perform, and the intermediate compound resulted in high atom economy. A bioassay indicated that some of the target compounds showed moderate to good insecticidal activity against P. xylostella and S. frugiperda larvae. Of these, compounds 6i, 6j, and 6n exhibited promising insecticidal activity against P. xylostella, while 6i, 6j, and 6t showed effective insecticidal activity against S. frugiperda. In particular, compound 6j displayed the best activity, which was superior to that of lead D-27. SAR analysis revealed that inserting an additional methylene spacer between the substituted phenyl ring and the amide bond can improve the insecticidal activity. Furthermore, molecular docking and protein verification demonstrated that 6j could act not only on EcR, but also on Of ChtI. The present results indicated that an appropriate increase in molecular flexibility could enhance the binding affinity of the compound with the EcR or Of ChtI, which would be very valuable for further structure optimization. This work provides a rational design strategy for the discovery of novel dual-target IGRs for green pest control.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules28093741/s1, Table S1: The price and manufacturers of the starting reagents; Table S2: Comparison of the yields of hexacyclic and heptacyclic intermediates; Table S3: Comparison of the yields of hexacyclic and heptacyclic compounds; Table S4: Elution gradient and analysis conditions of HPLC; Figure S1: Predicted binding modes of 6f and D-27 with EcR and Of ChtI; Figure S2: Predicted binding modes of 6b, 6j, and 6u with EcR and Of ChtI; Figure  S3: the HPLC chromatogram of 6j; physical and chemical properties, NMR, and HRMS spectra and data of 6a~6x.